Because of the feeding technology innovation, accelerated transport and communication facilities breeds of high performance breeds replaced local autochone breeds worldwide. These latter species however have an important role in gene conservation. Hungarian cold-blooded horse breeding stock are lacking pedigree, the actual founder breed mares are not known. For this reason, it is an major priority defining the genetic backround of the existing flock, for that breeding could operate with purposeful using of origin maternal founders. In the present study 195 cold-blooded Hungarian mares tail and mane sample were analized. Our analysis was carried out between 15531–15752 base pairs in mithocrondial DNA D-loop region, which reported a total of 222 base pairs. Fourtyone polymorphic sites were determined, which resulted in 39 haplotypes (h=39). The average pairwise differences were k=6.825. High haplotype and nucleotide diversity values were observed (Hd=0.968±0.003, π=0.026±0.003). Based on the previously defined variable positions of haplotypes defined by Jansen et al (2002), we groupped our haplotypes into haplogroups. 23 percent of the studied population (45 mares) belonged to haplogroup F1. Nearly 97% of the analyzed population was classified into one of eight haplogroups defined by Jansen.et al. (2002). This study gives genetic information nearly 25% of the Hungarian population. Another possibility would be patterning more mares or involving more genetic marker in the study which will assuming the possibility of a more comprehensive analysis.